APPLICANT'S DESCRIPTION: Retroviral vectors are currently the most popular gene delivery system used in human gene therapy clinical trials, but a major factor that would increase their in vivo efficacy would be the development of targeted vectors. To this end, we have been engineering the ecotropic murine leukemia virus (MuLV) envelope (Env) protein to bind to specific cell surface receptors and to thereby direct the vectors to specific target cells. Although modifications of the ecotropic Env protein have been reported that allow some degree of targeting to human cells, the low efficiency and general lack of utility of these methods has precluded their clinical use. Following the interaction with its natural receptor, the Env protein is thought to undergo a series of conformational changes that ultimately lead to the fusion of viral and host cell membranes. We have demonstrated that although engineered Env proteins can be shown to bind specifically to a targeted ligand, the subsequent fusion step is not triggered. In the Env proteins of different MuLV subtypes, distinct receptor binding regions exist within the framework of a more conserved protein scaffold that allow the use of different cellular receptors as targets. That such variation is naturally accommodated suggests that it will be possible to engineer the MuLV Env for targeted gene delivery, but that this will require detailed knowledge of the natural Env/receptor interactions. In this proposal, we describe our continuing studies to analyze the process of entry directed by the MuLV Env proteins. The availability of such information may enable us to take a more rational approach to the engineering of a targeted vector in the future. In addition, we propose to explore the of use targeting ligands to concentrate or 'tether' human-tropic MuLV Env proteins to defined target cells as an alternate approach to obtaining targeted gene delivery.